SELECTIVE ENRICHMENT OF GLYCOPROTEINS USING MAGNETIC MICROSPHERES AND THEIR SENSITIVE DETECTION WITH SURFACE ENHANCED RAMAN SPECTROSCOPY

Abstract

Glycoproteins play critical roles including cell−cell interaction; signal transduction, protein folding, molecular recognition and host-pathogen interactions in metabolism. Glycoproteins are also widely used in pharmaceutical industry because of effectiveness in drug stability, pharmacokinetic and immunogenicity. Antibodies and antibody-derived molecules are the leading family of biopharmaceuticals. One of the most popular antibody is Immunoglobulin G (IgG) and its production at high levels of purity is an important process necessity. In the first part of this study, a magnetic silica based sorbent was used to purify IgG glycoprotein from complex serum media. The sorbent was functionalized with amino phenyl boronic acid (APBA) group to obtain pH-dependent diol-sensitive system for IgG purification. Adsorption-desorption mechanism was observed and optimum conditions were studied. 0.05 M HEPES buffer, pH 8.5, was used as binding buffer. IgG adsorption behavior was found compatible with Langmuir model; it shows that adsorption has occurred as a single layer and homogeneously. Maximum adsorption capacity of IgG was calculated as 84 mg.g-1. After the adsorption process, optimization of the desorption media was performed. Optimum isolation of IgG was obtained with 5 mg sorbent in 1 M Tris-Cl (pH 8.5) with 0.2 M NaCl. After the purification process, IgG in serum was concentrated 4-fold and IgG was found approximately 80% purity in elution buffer. In the second part of this study, after the effective purification step of glycoproteins, sensitive and fast detection of these glycan structures were investigated for possible use in pathogen detection, early diagnosis of today’s important disorders like cancer, diabetes and effective pharmaceuticals production. For this purpose, Surface Enhanced Raman Spectroscopy (SERS) technique was used with diol sensitive mercapto phenyl boronic acid (MPBA) functionalized Ag nanoshell coated magnetic polymethyacrylate microspheres (MPBA-Ag@MagPMMS). As model proteins, much similar RNase A protein (negative control) and RNase B glycoprotein were used with this special sandwich system. Characterization and quantification of adsorbed glycoproteins were realized with this technique. RNase B concentrations down to 0.1 ng/mL, nearly 10-15 M, could be detected with this method effectively.